Elson H F, Gentry M K, Doctor B P
Department of Biological Chemistry, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
Biochim Biophys Acta. 1992 Dec 8;1156(1):78-84. doi: 10.1016/0304-4165(92)90099-g.
Cell-bound acetylcholinesterase (AChE) was found to be an early differentiation marker on embryonic chick skeletal myoblasts in mixed primary cell cultures. AChE biosynthesis was detected and characterized by (a) a sensitive microtiter assay, (b) use of selective inhibitors, and (c) with mono- and polyclonal antibodies. Both secreted and cell-bound AChE appeared on the first day in culture, at a time when no muscle cell fusion was observed. Characterization of this enzyme revealed that true AChE was bound and secreted by myoblasts. BW284c51, which permeates cell membranes poorly, inhibited all the cell-associated AChE activity on myoblasts, suggesting that the activity measured was on the outer cell surface. On the other hand, fibroblasts appeared to have no or very little bound enzyme and the low level of secreted enzyme activity had the characteristics of pseudo-, or butyrylcholinesterase. Polyclonal anti-Torpedo californica electroplax AChE antibody and several monoclonal antibodies were found to bind specifically to chick myoblasts. Since the cells had not been made permeable before antibody binding, a membrane-bound form of the enzyme was most likely being detected. The cell-bound true AChE was present in identifiable quantities from the first day of culture. Membrane-bound AChE can thus serve as an early differentiation marker for embryonic chick myoblasts in mixed primary cultures.
在混合原代细胞培养物中，细胞结合型乙酰胆碱酯酶（AChE）被发现是胚胎鸡骨骼肌成肌细胞上的一种早期分化标志物。通过（a）灵敏的微量滴定法、（b）使用选择性抑制剂以及（c）使用单克隆和多克隆抗体对AChE生物合成进行了检测和表征。分泌型和细胞结合型AChE在培养的第一天就出现了，此时未观察到肌肉细胞融合。对这种酶的表征显示，真正的AChE由成肌细胞结合并分泌。BW284c51对细胞膜的渗透性较差，它抑制了成肌细胞上所有与细胞相关的AChE活性，这表明所测量的活性位于细胞外表面。另一方面，成纤维细胞似乎没有或只有很少的结合酶，并且分泌型酶活性的低水平具有假性或丁酰胆碱酯酶的特征。发现多克隆抗加州电鳐电板AChE抗体和几种单克隆抗体能特异性结合鸡成肌细胞。由于在抗体结合之前细胞没有被通透处理，很可能检测到的是酶的膜结合形式。从培养的第一天起，细胞结合型真正的AChE就以可识别的量存在。因此，膜结合型AChE可作为混合原代培养物中胚胎鸡成肌细胞的早期分化标志物。
